Overexpression of AcWRKY31 Increases Sensitivity to Salt and Drought and Improves Tolerance to Mealybugs in Pineapple

Pineapple is a globally significant tropical fruit, but its cultivation faces numerous challenges due to abiotic and biotic stresses, affecting its quality and quantity. WRKY transcription factors are known regulators of stress responses, however, their specific functions in pineapple are not fully understood. This study investigates the role of AcWRKY31 by overexpressing it in pineapple and Arabidopsis. Transgenic pineapple lines were obtained using Agrobacterium-mediated transformation methods and abiotic and biotic stress treatments. Transgenic AcWRKY31-OE pineapple plants showed an increased sensitivity to salt and drought stress and an increased resistance to biotic stress from pineapple mealybugs compared to that of WT plants. Similar experiments in AcWRKY31-OE, AtWRKY53-OE, and the Arabidopsis Atwrky53 mutant were performed and consistently confirmed these findings. A comparative transcriptomic analysis revealed 5357 upregulated genes in AcWRKY31-OE pineapple, with 30 genes related to disease and pathogen response. Notably, 18 of these genes contained a W-box sequence in their promoter region. A KEGG analysis of RNA-Seq data showed that upregulated DEG genes are mostly involved in translation, protein kinases, peptidases and inhibitors, membrane trafficking, folding, sorting, and degradation, while the downregulated genes are involved in metabolism, protein families, signaling, and cellular processes. RT-qPCR assays of selected genes confirmed the transcriptomic results. In summary, the AcWRKY31 gene is promising for the improvement of stress responses in pineapple, and it could be a valuable tool for plant breeders to develop stress-tolerant crops in the future.


Introduction
Plant breeders are currently facing a huge challenge developing new, high-yielding, and stress-tolerant crop varieties that can cope with environmental cues.Plants encounter various stresses throughout their life cycle, such as salinity, drought, temperature fluctuations, and pathogen infections [1].To adapt to challenging environments, plants attempt to regulate their physiological and biochemical mechanisms in a timely manner [2,3].At the same time, plant breeders are working on the development of stress-tolerant varieties to meet global demand.
The detrimental effects of abiotic stress on plants primarily arise from unfavorable alterations in the natural environment due to changes in temperature, salinity, etc.These changes promote corresponding physiological and biochemical responses in plants, ultimately causing significant harm to their inherent traits and phenotypic characteristics, leading to crop losses [4].High levels of plant osmotic pressure and high levels of salinity can impair plant growth and development [5].Drought is another significant factor that exerts detrimental effects on plant processes [6].Additionally, drought stress has been associated with increased plant root biomass [7].When the water content decreases in plant environments, the water potential in leaves also declines, which impairs photosynthesis and metabolism, and, in severe cases, leads to plant mortality [8].Biotic stress, primarily caused by harmful pathogens, can penetrate plant cuticles, invade stomata, or exploit natural wounds to disrupt normal organ functions, leading to reduced photosynthetic rates, water imbalances, and nutrient transport obstacles [9,10].
Ananas comosus (L.) Merr.(pineapple) is an economically important crop that is distinctly exposed to disease conditions caused by bacteria, fungi, viruses, and nematodes, as well as abiotic stresses, leading to yield reductions [11].Dysmicoccus brevipes (pineapple mealybug) is one of the most serious pests, and it weakens plants by sap-sucking and acts as a vector for the pineapple mealybug wilt-associated virus (PMWaV) [12].Another destructive pathogen, Sclerotinia sclerotiorum, infects over 400 plant species, impacting crop yield and quality [13].These pathogens pose a significant threat to various plants, including dicotyledons, such as sunflower, soybean, and rape, as well as monocotyledons, like onion and tulip [14].Consequently, advanced biotechnologies play an essential role in the improvement of plant resistance to environmental stresses, identifying stress-resistant genes and facilitating the development of new plant varieties.
Among the transcription factor (TF) families involved in stress regulation, WRKY TFs are known for their diverse regulatory mechanisms.Typical WRKY proteins efficiently bind to W-box elements to modulate downstream gene transcription and can form protein complexes by interacting with other activated elements, enhancing their transcriptional binding ability.Structurally, WRKY TFs consist of two main domains: the DNA-binding N-terminal domain and the C-terminal zinc finger structure [15].The WRKY DNA binding domain exhibits variations within the family according to the basic features of conservative heptapeptides, such as WRKYGMK, WRKYGKK, WRKYGQK, WSKYGQK, WKRYGQK, WVKYGQK, and WKKYGQK [16].Zinc finger structures are primarily composed of C 2 H 2 and C 2 HC motifs and are crucial constituents of WRKY TFs.WRKY TFs are classified into subfamilies I, II, and III by the number of their domains and their zinc finger-like structure [17].The data from an evolutionary analysis showed that subfamily II can divide into sub-subfamilies, like IIa, IIb, IIc, IId, and IIe [18].Furthermore, some WRKY families also contain the proline enrichment domain, glutamate enrichment domain, and a leucine structure [19].
The role of WRKY TFs in regulating responses to abiotic stress has been studied in various species.The IbWRKY47 gene is upregulated in salt stress and confers salinity resistance to sweet potatoes [20].MiR156/SPL induces the expression of the salt-tolerant gene MdWRKY100, improving the salt tolerance of Begonia [21].SbWRKY50 participates in sweet sorghum plant responses to salt stress by controlling its ion balance according to the binding of the upstream promoters of AtSOS1 and AtHKT1 [22,23].In chrysanthemum, CmWRKY17 is negatively regulated by salt stress [24].Transcripts, proteins, metabolite levels, hormones, ROS, small RNAs, epigenetic modifications, post-translational modifications, and environmental cues are the main factors regulating the seed germination of Arabidopsis and rice [25].GhWRKY68 regulates the ABA-mediated pathway and reduce resistance to drought and salt in cotton [26].The overexpression of the BdWRKY36 transgenic line can reduce ROS accumulation by the activation of NtNCED1, NtDREB3, and NtLEA5 in the ABA biosynthesis pathway and lead to significant resistance to drought stress in tobacco [27].In pepper, CaWRKY6 activates CaWRKY40 and makes it more resistant to heat and humidity stress [28].GmWRKY27 inhibits the GmNAC29 promoter through an independent inhibitory effect and inhibits the expression of GmNAC29 with GmMYB174 to improve plant resistance to salt and drought stress [29].In pineapple, 54 WRKY genes have been investigated in this family [30].However, there has been less research regarding functional analyses of the pineapple WRKY family's genes.Huang et al. reported that the ectopic overexpression of AcWRKY31 negatively affects tolerance to drought and salt stresses in rice [31].The first-ever study conducted with transgenic pineapple showed the ability of overexpressed AcWRKY28 in pineapple to confer salt stress tolerance [32].Through the regulation of bromelain and oxidative stress, the effect of the DA-6 and COS PGRs can create a tolerance to drought in pineapple [33].
Ecological stresses have a significant impact on the fruit development, quality, and yield of pineapple plants [34].Conventional pineapple breeding for new variety development is time-consuming for commercial production, and self-incompatibility further complicates this process.Molecular breeding and biotechnological tools make breeding programs easier and more efficient.Transgenic AcWRKY31-OE pineapple and Arabidopsis plants were successfully developed using the Agrobacterium-mediated transformation method.The results of this study showed that the overexpression of AcWRKY31 increased sensitivity to drought and salt tolerance in both pineapple and Arabidopsis and increased tolerance to biotic stress caused by pineapple mealybugs in pineapple.In Arabidopsis, the overexpression of AcWRKY31 increased resistance to Sclerotinia sclerotiorum infection.The findings of our study on stress-responsive genes provide insight into molecular breeding for developing improved pineapple varieties to boost the economic growth of producers.

The Expression Profiles and Subcellular Localization of AcWRKY31
By analyzing the gene expression patterns, we can preliminarily identify the developmental processes involved and provide a theoretical foundation for subsequent phenotypic and functional analyses.To examine the expression patterns of AcWRKY31 in different pineapple tissues, we downloaded and analyzed the transcriptome data from various tissues, including root, leaf, flower, calyx, petal, stamen, pistil, ovule, and fruit tissues [35,36].The expression level of AcWRKY31 was shown to be the highest in the pistil, followed by the ovule and root (Figure 1A).In addition, an RT-qPCR analysis confirmed the accuracy of the expression level of AcWRKY31 in different pineapple tissues and revealed high expression levels in the pistil and ovule (Figure 1B).Based on the tobacco transient transformation, we found that AcWRKY31 is localized in the nucleus (Figure 1C).These expression patterns suggest that AcWRKY31 serves an important function in pineapple pistil and ovule development as well as in root development for water absorption.

Phylogenetic Analysis and Sequence Alignment of AcWRKY31
To characterize the pineapple gene AcWRKY31, the CDS sequence and genome sequence were downloaded, and the gene structure is depicted using GSDS 2.0 (Figure 2A).A phylogenetic tree of the pineapple AcWRKY31 and its homologous genes in Arabidopsis and rice was constructed.The phylogenetic tree indicated that AcWRKY31 shares a high degree of similarity with AtWRKY53 in Arabidopsis and OsWRKY113 in rice (Figure 2B).In addition, the alignment of multiple protein sequences showed that AcWRKY31 belongs to group III of the pineapple WRKY TF family, which contains a conserved WRKYGQK domain and C 2 HC-type zinc finger motif (C-X7-C-X23-H-X-C) (Figure 2C).To characterize the pineapple gene AcWRKY31, the CDS sequence and genome sequence were downloaded, and the gene structure is depicted using GSDS 2.0 (Figure 2A).A phylogenetic tree of the pineapple AcWRKY31 and its homologous genes in Arabidopsis and rice was constructed.The phylogenetic tree indicated that AcWRKY31 shares a high degree of similarity with AtWRKY53 in Arabidopsis and OsWRKY113 in rice (Figure 2B).In addition, the alignment of multiple protein sequences showed that AcWRKY31 belongs to group III of the pineapple WRKY TF family, which contains a conserved WRKYGQK domain and C2HC-type zinc finger motif (C-X7-C-X23-H-X-C) (Figure 2C).

Transformation and Identification of Overexpressed AcWRKY31 in Pineapple
To investigate the regulations and functions of AcWRKY31, a genetic transformation was performed using an Agrobacterium-mediated transformation system as described in Priyadarshani et al. with a few modifications [37].Transgenic callus and plants were selected rigorously (Figure 3A-G) in the selection medium.The screened plants were further confirmed through PCR testing and sequencing.A total of 12 independent positive AcWRKY31-overexpressed pineapple plants were verified (Figure 3H).The WT plants and 12 AcWRKY31-OE transgenic pineapple plants were used to extract the total RNA, and RT-qPCR testing was performed to confirm the expression levels of AcWRKY31.The result showed that the expression level of AcWRKY31 was increased more than twice in all of the overexpressed transgenic plants compared to the wild-type plants (Figure 3I).

Transformation and Identification of Overexpressed AcWRKY31 in Pineapple
To investigate the regulations and functions of AcWRKY31, a genetic transformation was performed using an Agrobacterium-mediated transformation system as described in Priyadarshani et al. with a few modifications [37].Transgenic callus and plants were selected rigorously (Figure 3A-G) in the selection medium.The screened plants were further confirmed through PCR testing and sequencing.A total of 12 independent positive AcWRKY31-overexpressed pineapple plants were verified (Figure 3H).The WT plants and 12 AcWRKY31-OE transgenic pineapple plants were used to extract the total RNA, and RT-qPCR testing was performed to confirm the expression levels of AcWRKY31.The result showed that the expression level of AcWRKY31 was increased more than twice in all of the overexpressed transgenic plants compared to the wild-type plants (Figure 3I).

Overexpression of AcWRKY31 Reduces Tolerance to Salt and Drought Stresses in Pineapple
To investigate the function of AcWRKY31 in pineapple, AcWRKY31-OE transgenic plants were subjected to salt and drought stress.After three weeks of treatment with 400 mM NaCl and 400 mM mannitol solutions, the leaves of the AcWRKY31-OE pineapple plants were wilted, dried, and drooped, while the leaves of the wild-type (WT) pineapple plants remained upright (Figure 4A,B).After undergoing one month of recovery in a greenhouse, the AcWRKY31-OE transgenic pineapple plants were completely dried and noticeably lighter compared to the WT plants.According to the results of the RT-qPCR analysis, enzyme-and salt-stress-related marker genes, such as AcPOD1, AcABI5, AcABA1, and AcPR1 genes, in the AcWRKY31-OE plants showed reduced relative expression levels compared to those of the WT plants (Figure 4C).Similarly, the relative expression levels of hormone-and drought-stress-related marker genes (AcCAT1, AcPR1, AcLOX4, AcABA1, AcRD22, and AcDREB2A) in the AcWRKY31-OE plants were lower than then those to the WT pineapple (Figure 4D).These findings indicate the overexpression of AcWRKY31 enhances the sensitivity to salt and drought stress in pineapple.
Plants 2024, 13, x FOR PEER REVIEW 7 of 24 To investigate the function of AcWRKY31 in pineapple, AcWRKY31-OE transgenic plants were subjected to salt and drought stress.After three weeks of treatment with 400 mM NaCl and 400 mM mannitol solutions, the leaves of the AcWRKY31-OE pineapple plants were wilted, dried, and drooped, while the leaves of the wild-type (WT) pineapple plants remained upright (Figure 4A,B).After undergoing one month of recovery in a greenhouse, the AcWRKY31-OE transgenic pineapple plants were completely dried and noticeably lighter compared to the WT plants.According to the results of the RT-qPCR analysis, enzyme-and salt-stress-related marker genes, such as AcPOD1, AcABI5, AcABA1, and AcPR1 genes, in the AcWRKY31-OE plants showed reduced relative expression levels compared to those of the WT plants (Figure 4C).Similarly, the relative expression levels of hormone-and drought-stress-related marker genes (AcCAT1, AcPR1, AcLOX4, AcABA1, AcRD22, and AcDREB2A) in the AcWRKY31-OE plants were lower than then those to the WT pineapple (Figure 4D).These findings indicate the overexpression of AcWRKY31 enhances the sensitivity to salt and drought stress in pineapple.

Overexpression of AcWRKY31 Increases the Resistance to Pineapple Mealybug
To investigate the function of AcWRKY31 that response to biotic stress, four-monthold AcWRKY31-OE and WT pineapple plants were inoculated with Dysmicoccus brevipes.There were no significant differences in AcWRKY31-OE and WT plants in the first two months after inoculation.However, after 3 months of inoculation, we observed a signifi-

Overexpression of AcWRKY31 Increases the Resistance to Pineapple Mealybug
To investigate the function of AcWRKY31 that response to biotic stress, four-month-old AcWRKY31-OE and WT pineapple plants were inoculated with Dysmicoccus brevipes.There were no significant differences in AcWRKY31-OE and WT plants in the first two months after inoculation.However, after 3 months of inoculation, we observed a significantly higher density of colonies on the crown meristem and dorsal surfaces of the WT leaves compared to that of those the AcWRKY31-OE pineapple plants.In addition, the size of the colonies was significantly larger in the WT plants (Figure 5A).In the WT plants, there were approximately 200 colonies on the dorsal surface and less than 100 colonies on the ventral surface of the pineapple leaves, and the smallest colony sizes were dotted while the maximum colony diameter was 0.8 cm.In the transgenic pineapple leaves, the number of mealybug colonies did not exceed 60 on the dorsal surface and was less than 15 on the ventral surfaces.Most of the colonies were tiny, and the largest colonies were only 0.2 cm in diameter (Figure 5B,C).Interestingly, we found that the dorsal surface was more susceptible to mealybug infection in both the WT and transgenic plants compared to the ventral leaf surface.To understand the relationship between the higher infection rate on the dorsal side compared to the ventral side, free-hand transverse sections were cut, and the cellular arrangement was observed under a microscope.We found a soft epidermal cell layer and thick layers of water storage cells on the dorsal side of both the WT and transgenic leaves.On the ventral side of the leaves, rigid epidermal cells, chlorenchyma cells, fibers, and vascular bundles were observed (Figure 5D).According to the cell arrangements in both the WT and transgenic leaves, mealybugs easily penetrate the cell layers and infect the dorsal surfaces rather than the ventral surfaces.Based on the phenotypic characteristics and statistical analysis data of the colonies on the leaf surfaces, we can conclude that the overexpression of AcWRKY31 enhanced resistance to Dysmicoccus brevipes in the pineapple.

RNA-Seq Analysis of AcWRKY31-OE Pineapple
To study the functions and mechanisms of AcWRKY31 regulation in pineapple, a transcriptomic analysis was conducted by using the total RNA from the WT and AcWRKY31-OE transgenic pineapple.In this analysis, we identified 5357 upregulated genes and 662 downregulated genes from the transgenic pineapple plants compared to the WT plants.The putative functions of these differentially expressed genes (DEGs) are provided in Supplementary Tables S2.1 and 2.2.The Gene Ontology (GO) enrichment

RNA-Seq Analysis of AcWRKY31-OE Pineapple
To study the functions and mechanisms of AcWRKY31 regulation in pineapple, a transcriptomic analysis was conducted by using the total RNA from the WT and AcWRKY31-OE transgenic pineapple.In this analysis, we identified 5357 upregulated genes and 662 downregulated genes from the transgenic pineapple plants compared to the WT plants.The putative functions of these differentially expressed genes (DEGs) are provided in Supplementary Tables S2. 1 and S2.2.The Gene Ontology (GO) enrichment analysis of DEGs showed that 5357 upregulated genes were significantly enriched in the RNA metabolic process, tRNA aminoacylation, the establishment of localization in cell, intracellular transport, protein folding, RNA splicing via transesterification reaction, cellular response to stimulus, transcription according to RNA polymerase II, ncRNA processing, etc. (Figure 6A).The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the upregulated DEGs were mostly involved in the process of translation, protein kinases, peptidases and inhibitors, membrane trafficking, folding, sorting and degradation, endocytosis and translation factors, etc. (Figure 6B).Among the upregulated genes, a total of 30 genes were identified relating to diseases and pathogen response, and further analysis showed that 18 of them contained the conserved WRKY binding domain (W-box TGAC) in the promoter region.These 18 genes typically belong to the CC-NBS-LRR and TIR-NBS-LRR classes of the disease resistance protein family (Supplementary Table S2.3).The GO analysis of 662 downregulated genes revealed their involvement in 19 GO terms, primarily concentrated in transport, the establishment of localization, alpha-amino acid metabolic process, transmembrane transport and the organic substance catabolic process, the carbohydrate catabolic process, etc. (Figure 6C).In addition, the KEGG analysis pointed out that these downregulated genes were activated in seven pathways, especially in mitochondrial biogenesis, metabolism, glycosyltransferases, oxidative phosphorylation, protein families, signaling and cellular processes, energy metabolism, and transporters (Figure 6D).Furthermore, three differentially expressed genes (Aco010322: MYB-like transcription factor family protein, Aco014286: Disease resistance protein CC-NBS-LRR class family, and Aco024514: Tetratricopeptide repeat TPR-like superfamily protein; three that are downregulated (Aco001317: Mitochondrial substrate carrier family protein Aco002224: Beta-1,4-N-acetylglucosaminyltransferase family protein, and Aco010847: Glutaredoxin family protein from downregulated were selected and performed RT-qPCR analysis to confirm the accuracy of the RNA-sequencing data.The results showed that the expression profiles for these six genes are consistent and aligned with the RNA-seq data (Supplementary Figure S3).
Plants 2024, 13, 1850 9 of 22 like superfamily protein; three that are downregulated (Aco001317: Mitochondrial substrate carrier family protein Aco002224: Beta-1,4-N-acetylglucosaminyltransferase family protein, and Aco010847: Glutaredoxin family protein from downregulated were selected and performed RT-qPCR analysis to confirm the accuracy of the RNA-sequencing data.The results showed that the expression profiles for these six genes are consistent and aligned with the RNA-seq data (Supplementary Figure S3).

AcWRKY31-OE Transgenic Arabidopsis Reduces Tolerance to Salt and Drought Stresses
To confirm the heterologous functions of AcWRKY31-OE in Arabidopsis, we transformed the overexpressed vector 35S:AcWRKY31-GFP gene and its homologous gene 35S:AtWRKY53-GFP into Arabidopsis by the floral dip method.The overexpressed AcWRKY31 and its homologous gene (AtWRKY53) in Arabidopsis seeds underwent salt and drought treatments.Under the treatment of 100 and 150 mM NaCl, both the WT and transgenic Arabidopsis seeds could germinate completely; however, the fresh weight and root length of AcWRK31-OE Arabidopsis were significantly reduced compared to the WT plants (Figure 7A).Likewise, a notably adverse impact was observed for the results of the AcWRK31-OE Arabidopsis plants subjected to 200 mM and 250 mM mannitol treatments (Figure 7B).Furthermore, the same findings were observed in AtWRKY53-OE Arabidopsis under the stress of the salt and drought treatments (Supplementary Figures S4 and S5).Sun and Yu showed that the Atwrky53 mutant negatively regulates drought tolerance by mediating stomata movement [38].According to the phenotypic characters of this experiment, AcWRKY31 plays a negative role in the regulation of drought and salt stress response in Arabidopsis.To verify the further molecular mechanism of AcWRKY31-OE Arabidopsis in salt and drought stress, an RT-qPCR analysis was performed after the treatments.The expression levels of enzyme-related and stress-related genes, such as AtPOD1, AtLOX4, AtABI5, and AtPR1, were significantly decreased in all of the transgenic lines after the NaCl and mannitol treatments (Figure 7C,D).The results showed that the overexpression of AcWRKY31 enhanced the sensitivity to salt and drought stress in Arabidopsis.Based on the RT-qPCR assay of ABA stress treatment on WT pineapple, we can speculate that AcWRKY31 may be involved in the ABA-mediated stress response process.To prove this speculation, ABA stress treatments were performed in AcWRKY31-OE, AtWRKY53-OE, and in Atwrky53 Arabidopsis avn showed consistent findings that the ABA was induced in both overexpression lines (Supplementary Figure S6).AcWRKY31 may be involved in the ABA-mediated stress response process.To prove this speculation, ABA stress treatments were performed in AcWRKY31-OE, AtWRKY53-OE, and in Atwrky53 Arabidopsis avn showed consistent findings that the ABA was induced in both overexpression lines (Supplementary Figure S6).

AcWRKY31-OE Transgenic Arabidopsis Increases Resistance to the Sclerotinia sclerotiorum Pathogen
To investigate the function of AcWRKY31 in response to pathogenic infection, leaves of AcWRKY31-OE transgenic and WT Arabidopsis were inoculated with Sclerotinia sclerotiorum.After 24 h of infection, DAB staining was performed to observe the infected area, and the results revealed that the infected area of the WT leaves was significantly larger than that of three AcWRKY31-OE lines (Figure 8A,B).Disease-and hormone-related

AcWRKY31-OE Transgenic Arabidopsis Increases Resistance to the Sclerotinia sclerotiorum Pathogen
To investigate the function of AcWRKY31 in response to pathogenic infection, leaves of AcWRKY31-OE transgenic and WT Arabidopsis were inoculated with Sclerotinia sclerotiorum.After 24 h of infection, DAB staining was performed to observe the infected area, and the results revealed that the infected area of the WT leaves was significantly larger than that of three AcWRKY31-OE lines (Figure 8A,B).Disease-and hormone-related marker genes, such as AtICS1, AtPDF1.2,AtPR1, AtERF1, AtLOX4, and AtABI5, were selected for an RT-qPCR assay after infection [39][40][41].The RT-qPCR results showed the expression levels of these selected marker genes were significantly increased, indicating that the overexpression of AcWRKY31 enhanced the plants' sensitivity to pathogenic disease (Figure 8C).In addition, we inoculated the same pathogen on Atwrky53, Atwrky53-com, and Atwrky53pAcWRKY31 leaves.The affected areas of the Atwrky53pAcWRKY31 leaves were smaller than those of the Atwrky53 and Atwrky53-com, and the expression levels of marker genes were also increased in the Atwrky53pAcWRKY31 leaves (Figure 9A-C).Therefore, these results provided proof that the functions of AcWRKY31 and its homologous gene AtWRKY53 can increase the resistance to biotic stress in both pineapple and Arabidopsis.
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Discussion
Environmental factors such as drought, salt, pathogens, and pests have a significant impact on crop yield and quality.Pineapple, an important economic fruit crop, is susceptible to various ecological stresses during its lifespan.WRKY transcription factors (TFs) are involved in biotic and abiotic stress in different crops, such as pepper [42], cotton [43], chrysanthemum [24], and rice [44].In pineapple, 54 WRKY genes have been identified and reported by the authors of [30,34].However, only limited research has been conducted to elucidate the functions of WRKY genes in pineapple [31,32].Therefore, the current study was focused on cloning and transforming the AcWRKY31 gene to investigate its functions in pineapple.
WRKY transcription factors (TFs) can be divided into three groups (I, II, and III) based on their motifs and domains.Members of group III contain a conserved WRKYGQK domain and a C 2 HC zinc finger structure (C-X7-C-X23-H-X-C).Previous studies have shown that group III WRKY genes in Chinese rose [45], Sorghum [46], barley [47], maize [48], rice [49], Arabidopsis [50], and pineapple [30] share these same domains and motifs.In our study, AcWRKY31 exhibited high expression profiles and relative expression levels in the pistils and ovules (Figure 1A,B).The phylogenetic analysis and the alignment of multiple protein sequences revealed that the AcWRKY31 gene and its homologous genes in rice and Arabidopsis belong to the third subfamily from the WRKY transcription factor family, containing a conserved WRKYGQK domain and a C 2 HC zinc finger structure (C-X7-C-X23-H-X-C).Additionally, AcWRKY31 is exclusively localized in the nucleus and demonstrates transcriptional activation activity (Figure 2).Similarly, our expression profile findings of AcWRKY31 were consistent with the transcriptomic profile and displayed the same gene structure and functions.
In recent years, abiotic stresses, particularly salt and drought, have severely affected crops [51,52].Many researchers have investigated and reported on the role of WRKY genes in different crops.For instance, GhWRKY68 reduces resistance to salt and drought by affecting the germination rate, survival rate, and stomatal closure of transgenic tobacco.[26].AtWRKY53 is involved in drought stress and leaf senescence in Arabidopsis by mediating stomatal movement and ABA induction [38,42].In rice, OsWRKY46, OsWRKY64, and OsWRKY113 are upregulated in BR IRGA 409 and involve iron toxicity [53].Our current study demonstrated that AcWRKY31-OE transgenic pineapples exhibited adverse phenotypic characteristics compared to wild-type pineapples during drought and salt stress.The expression levels of enzyme-and stress-related marker genes, like AcPOD1, AcABI5, AcABA1, and AcPR1 for salt stress and AcCAT1, AcPR1, AcLOX4, AcABA1, AcRD22, and AcDREB2A for drought stress, were reduced in all transgenic pineapple lines compared to WT plants (Figure 4).In addition, the heterologous overexpression of AcWRKY31 in Arabidopsis also showed an increased sensitivity to salt and drought compared to the WT plants.Therefore, our findings indicate that the overexpression of AcWRKY31 enhances the sensitivity to abiotic stresses, especially salt and drought, in both pineapple and Arabidopsis.
Plants must adapt to drastic environmental changes throughout their lifespan by altering their biochemical and physiological pathways.Among those challenges, attacks from pathogens, insects, pests, and weeds can cause significant damage to plants and reduce crop yields.Many researchers have reported the effects of WRKY TFs on different biotic stresses, for example, the overexpression of AtWRKY28 conferred resistance to Sclerotinia and Botrytis cinerea in Arabidopsis [54], the overexpression of AtWRKY75 promoted resistance to Sclerotinia and Pseudomonas syringae [55], and AtWRKY70 Arabidopsis increased the resistance to SA-mediated powdery mildew but rendered plants sensitive to JA-mediated black spot [56].The AtWRKY22 genes promote susceptibility to aphids and modulate salicylic acid and jasmonic acid signaling [57].In pineapple, Zhou et al. stated that AcWRKY28 mediated the activation of AcCPK genes and conferred drought and salt stress tolerance in transgenic pineapple and resistance to Sclerotinia in transgenic Arabidopsis [32].In rice, OsWRKY6 positively regulated resistance to Xanthomonas oryzae [58].The overexpression of OsWRKY31 and OsWRKY13 enhanced the resistance to rice blast in rice [59,60].The pineapple mealybug (Dysmicoccus brevipes) is one of the most common insects that affect plant quality and yield by sap-sucking and acts as a vector for the pineapple mealybug wiltassociated virus (PMWaV) [12].Our result showed that the overexpression of AcWRKY31 significantly enhances the resistance to mealybugs and Sclerotinia sclerotiorum infection.The RT-qPCR analysis of disease-related and hormone-related marker genes (AtICS1, AtPDF1.2,AtPR1, AtERF1, AtLOX4, and AtABI5) also indicated that AcWRKY31 improved pineapple tolerance to biotic stress.
The WRKY family is also considered to play a crucial role in hormone signaling pathways, including ABA, BRs, ETH, JA, and SA, and can participate in plant defenses [61,62].The ABA signaling pathway is mainly involved in the regulation of the abiotic stress response in plants [63].The nuclear protein GmWRKY12 was responsive to drought, salt, ABA, and salicylic acid (SA) stress [64].The overexpression of GhWRKY1 in Arabidopsis constitutively activated ABA biosynthesis genes, signaling genes, responsive genes, and drought-related maker genes and led to an enhanced tolerance to drought [65].Under an exogenous hormone treatment, the expression level of AcWRKY31 was reduced at 4 h, 8 h, and 12 h, then it instantly increased at 24 h and 48 h under an ABA treatment (Supplementary Figure S1).In addition, the phenotype of AcWRKY31-OE Arabidopsis lines in the ABA treatment showed a significantly reduced resistance to ABA than WT plants.During the salt and drought stress experiments, it was found that the expression levels of marker genes in ABA-related signaling pathways were significantly increased.This expression change may be due to the involvement of AcWRKY31 in the ABA signaling pathway, which coordinates metabolic and physiological processes within plants and leads to changes in gene expression to adapt to alternative environments, but further research is needed.Therefore, we speculate that AcWRKY31 may regulate plant stress response through ABAmediated signaling pathways.
Altogether, based on transcriptome and experimental results, we conclude that AcWRKY31 plays distinct roles in pineapple under both biotic and abiotic stress.Its reduction in plant response to drought and salt stress may be mediated by the ABA signaling pathway, while its increase in plant resistance to biological stress may be achieved by regulating the expression of disease-resistance-related genes (Figure 10).

Plant Materials and Pineapple Callus Induction
The pineapple (Tainong 11) plants and Arabidopsis (Columbia-0) seeds used in this experiment were obtained from Qin Lab, Center of Genomics Biotechnology, Fujian Agriculture and Forestry University, Fujian, China, where the experiments took place.For pineapple callus induction, we used the pineapple micro-propagation methods of Priyadarshani [66] with some modifications, especially for sterilization processes.Briefly, the explants such as leaf bases and crown meristems were soaked in disinfectant solution overnight to reduce contaminations and then washed with running tap water.In addition, the explants were surface sterilized with 75% ethanol for 1 min and then sterilized with 3-5% sodium hypochlorite solution for 8-10 min.Furthermore, the explants were cut into sizes of 0.5-1.5 cm and then sterilized with 0.2% of HgCl2 solution for 10 min.The explants were washed with sterilized double-distilled water 3-5 times after each sterilization.After sterilizations, the samples were air-dried and cultured in full MS solid medium (with pH of −5.8) containing 30% sucrose, 4 mg/L of BAP, and 0.2 mg/L of NAA for callus induction.The callus was subcultured in full MS solid medium containing 1 mg/L of BAP and 0.2 mg/L of NAA.All cultures were kept in a tissue culture room under the following controlled conditions: 8 h/16 h dark and light cycle, light intensity of 3000 lux, and temperature of ±26 °C.All the sterilization and cultural processes were carried out in an aseptic condition.

Plant Materials and Pineapple Callus Induction
The pineapple (Tainong 11) plants and Arabidopsis (Columbia-0) seeds used in this experiment were obtained from Qin Lab, Center of Genomics Biotechnology, Fujian Agriculture and Forestry University, Fujian, China, where the experiments took place.For pineapple callus induction, we used the pineapple micro-propagation methods of Priyadarshani [66] with some modifications, especially for sterilization processes.Briefly, the explants such as leaf bases and crown meristems were soaked in disinfectant solution overnight to reduce contaminations and then washed with running tap water.In addition, the explants were surface sterilized with 75% ethanol for 1 min and then sterilized with 3-5% sodium hypochlorite solution for 8-10 min.Furthermore, the explants were cut into sizes of 0.5-1.5 cm and then sterilized with 0.2% of HgCl 2 solution for 10 min.The explants were washed with sterilized double-distilled water 3-5 times after each sterilization.After sterilizations, the samples were air-dried and cultured in full MS solid medium (with pH of −5.8) containing 30% sucrose, 4 mg/L of BAP, and 0.2 mg/L of NAA for callus induction.The callus was subcultured in full MS solid medium containing 1 mg/L of BAP and 0.2 mg/L of NAA.All cultures were kept in a tissue culture room under the following controlled conditions: 8 h/16 h dark and light cycle, light intensity of 3000 lux, and temperature of ±26 • C. All the sterilization and cultural processes were carried out in an aseptic condition.

Vector Construction, Gene Transformation of AcWRKY31, and Transgenic Plant Selection in Pineapple
The peptide sequences of AcWRKY31 (Aco000358.1) and its homologous genes from rice were downloaded from the Phytozome13 database "https://phytozome-next.jgi.doe.gov/ (accessed on 20 September 2021)" and those of Arabidopsis were downloaded from TAIR "https://www.arabidopsis.org/(accessed on 20 September 2021)".The CDS sequence (containing 1098 bps) and genome sequence (containing 2068 bps) of AcWRKY31 were downloaded from the Phytozome13 database, and the gene structure was displayed by GSDS 2.0.The protein sequences of AcWRKY31 and its two homologous genes from rice and Arabidopsis were used, and the alignment of multiple sequences was carried out using DNAMAN V6.0 software.The specific primers were designed using SnapGene TM 1.1.3software.The total RNA from young pineapple leaves was extracted and cloned to the cDNA.The cDNA and gene-specific primers of AcWRKY31 were used for PCR amplification to obtain the target DNA.After purification of the PCR products, the target DNA was cloned into the pENTER/D-TOPO vector and then recombined into the final pGWB502 vector using LR Clonase II enzyme mix (Thermo Fisher Scientific, Waltham, MA USA).The plasmids from the final vectors were extracted and transformed into Agrobacterium (GV3101) strains.The Agrobacterium with targeted genes was transformed into pineapple callus.In the transformation process, positive pineapple callus selection and plant screening were carried out as explained by Priyadarshani [37,67] with some modifications, especially in regards to selection time.Time for selection and antibiotic concentration were optimized according to the gene.Three repeated selections were carried out alternatively in the selection and normal medium.Following selection, the resilient plantlets were excised and cultivated on an MS solid medium supplemented with 0.2 mg/L NAA to stimulate root growth.Putative transgenic plants underwent DNA extraction for subsequent PCR amplification.DNA samples yielding positive PCR outcomes were forwarded to Bosun Biotechnology (Shanghai, China) Co., LTD for sequencing.

Analysis of Salt and Drought Stress in AcWRKY31-Overexpressed Pineapple
To analyze the response of transgenic and wild-type (WT) pineapple plants to salt and drought stress, plantlets at comparable growth stages were selected for experimentation.WT and transgenic pineapple plantlets were transplanted into pots with composted soil and placed in a greenhouse at 30 • C ± 2 • C for four months until they grew into adult pineapple plants.After four months, six AcWRKY31-OE transgenic plants and WT pineapple plants were treated with 100 mL of 400 mM NaCl solution for salt stress and 400 mM mannitol solution for drought stress.The phenotypic characteristics were noted and recorded daily.In addition, leaf samples of treated WT and AcWRKY31-OE transgenic pineapple were collected at 0 h, 12 h, and 24 h, respectively.After irrigating for 3 weeks, the phenotypic characters of treated WT and transgenic pineapple plants were compared.For the molecular assays, the total RNA was isolated from these treated samples and reverse-transcribed into cDNA for further experiments.Selected marker genes were analyzed using RT-qPCR with specific primers (Supplementary Table S1).

Analysis of Biotic Stress in AcWRKY31-OE Transgenic Pineapple
To investigate the role of AcWRKY31 in biotic stress response, pineapple mealybugs (Dysmicoccus brevipes) from a previously infected pineapple plant in the greenhouse were used to inoculate the wild-type (WT) and transgenic pineapple plants.A known number of mealybugs were inoculated onto the leaves of four-month-old, healthy wild-type (WT) and transgenic pineapple plants.Plants were maintained in the greenhouse at 30 • C ± 2 • C and were observed weekly.After four months, disease symptoms, plant growth, and insect colonization were visually inspected and recorded.The statistical data of mealybugs colonies were counted and calculated using IBM SPSS Statistics 24.0.0 software.

Genetic Transformation of AcWRKY31 and AtWRKY53 in Arabidopsis
To confirm and validate the functions of AcWRKY31, we transformed AcWRKY31, its homologous gene AtWRKY53, and knockoff mutant Atwrky53 into Arabidopsis using the floral dip method [68].Inflorescences were immersed in Agrobacterium solution containing the targeted plasmids: p35S:AcWRKY31 and p35S: AtWRKY53 [68,69].Subsequently, the infected plants were allowed to grow until the seeds were ready for harvesting.The seeds from the T 0 generation were germinated in soil and selected by spraying them with Basta (1:1000 dilution of water) to obtain transgenic plants.DNA was extracted from selected putative plants by using the CTAB method [70] to identify the positive plants for T 1 generation through PCR.The second-generation seeds were obtained by repeated screening and identification, and they were used for subsequent studies.

Analysis of Abiotic Treatments in Transgenic Arabidopsis
The previous study reported that the expression of WRKY TFs in pineapple can be affected by temperature stresses (4 • C and 45 • C), drought, and salt stresses [31].To understand the regulation of heterologous overexpression of AcWRKY31 under abiotic stress, the wild-type and T 2 generation of transgenic Arabidopsis seeds were treated with 100 mM-150 mM of NaCl for salt stress, 250 mM-300 mM of mannitol for drought stress, and 0.25-0.75µM of ABA for ABA stress.After 7 days, the germination rates, fresh weight, and root length were investigated.To analyze the expression level of AcWRKY31, RT-qPCR analysis was performed for salt and drought stress with stress-related marker genes.All the experiments were conducted with three replicates.

Analysis of Sclerotinia sclerotiorum Inoculation on Transgenic Arabidopsis
To examine the functions of AcWRKY31 in response to pathogens, WT and three independent lines of AcWRKY31-OE, AtWRKY53-OE, Atwrky53, Atwrky53-com, and Atwrky53-AcWRKY31 T 2 -generation Arabidopsis seeds were used.The preserved fungal strain of Sclerotinia sclerotiorum was sub-cultured on potato dextrose agar medium for the production of new hyphae.Briefly, five seeds from each line were planted in plastic pots with composted soil for germination.Leaves from 3-week-old transgenic and WT Arabidopsis plants were inoculated with the same quantity of Sclerotinia sclerotiorum.The infected area was analyzed after 24 h with DAB staining to analyze the accumulation of reactive oxygen species.For DAB staining, leaves were immersed in 50 mL of DAB aqueous solution (0.05 g DAB; ddH 2 O 50 mL) overnight in darkness.Dyed leaves were de-colorized using 75% ethanol in a water bath for 15 min.The lysis areas were recorded by taking photographs and measured using ImageJ 1.51j/Java 1.8.0-112(64-bit) software [71].The data were analyzed using the Student's t-test and GraphPad Prism 8.3.0 (538).All the experiments were repeated three times.

Analysis of Transcriptomic Data
Total RNA was extracted from three biological replicates of wild-type (WT) and AcWRKY31-overexpressing (AcWRKY31-OE) pineapple leaves using the Trizol method (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.Raw reads and adapter sequences were processed to remove ambiguities and adapters using TRIM-MOMATIC v 0.3 software [72].The RNA libraries were sequenced by the Novogene company on a HiSeq2500 sequencing instrument using 150 bp paired-end protocols.The processed reads were aligned to the reference genome using Tophat v2.1.1 software with default parameters.Then, the transcripts were quantified and assembled according to Cufflinks, and the differently expressed genes (DEGs) were identified.Differential ex-pression was determined based on a log2 fold change −1≤ and ≥+1 and a value of FDR ≤ 0.05) [73].Kyoto Encyclopedia of Genomes and Genes (KEGG) and Gene Ontology (GO) analyses of DEGs were carried out using TBtools v1.09 software [74] and R package UpSet v1.0.0.The promoter region analysis was performed through the online PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)(accessed on 13 March 2024).RT-qPCR analysis of six selected genes from up-and downregulated genes was performed to actuate the function of AcWRKY31 with the RNA-Seq data.The raw data sequence in Fastq format was deposited in the China National Center for Bioinformation Database (https://bigd.big.ac.cn;BioProject ID: PRJCA019756) (accessed on 13 September 2023).

Quantitative Real-Time PCR Analysis
The cDNA was synthesized from extracted total RNA according to the instructions of AMV reverse-transcriptase kit (Takara, Shiga, Japan) [75].RT-qPCR was carried out using SYBR Premix Ex Taq II (Takara, Japan) system and Bio-Rad Real-Time PCR system.The reaction was conducted in a total volume of 20 µL with 10 µL of enzyme mix, 7.5 µL of nuclease-free water, 1.5 µL of cDNA, and 0.5 µL of each specific forward and reverse primer.The RT-qPCR was performed by using given conditions (95 • C-30 s, 95 • C-5 s, 60 • C-34 s, and 95 • C-15 s) with 40 cycles [76].The experiment was performed in triplicate.According to the comparative threshold period (2 −∆∆Ct ) protocol, the level of relative expression in stress-related genes was analyzed and confirmed [77].

Conclusions
The outcomes of our current research highlight the notable expression patterns of AcWRKY31, particularly in the pistil and ovule of pineapple.Notably, our investigations reveal that the upregulation of AcWRKY31 in both pineapple and Arabidopsis plants is correlated with a reduced tolerance to salt and drought stress, alongside a heightened resistance to pests and pathogens.These findings underscore the intricate response mechanisms of pineapple to diverse stressors.Consequently, our study yields significant insights crucial for the development of advanced biotechnological interventions and breeding approaches aimed at enhancing the resilience and productivity of pineapple varieties.

Figure 1 .
Figure 1.Expression levels of AcWRKY31 in different parts of pineapple tissues; (A) Expression profiles of RNA-sequencing; (B) Relative expression levels AcWRKY31 from RT-qPCR analysis.The error bars indicate ±SD (n = 3) (C) Subcellular localization of AcWRKY31 that located in nucleus (Bar = 50 µm; Notes: blue and green color dots indicate the nucleus of cells under microscope).

Figure 1 .
Figure 1.Expression levels of AcWRKY31 in different parts of pineapple tissues; (A) Expression profiles of RNA-sequencing; (B) Relative expression levels AcWRKY31 from RT-qPCR analysis.The error bars indicate ±SD (n = 3) (C) Subcellular localization of AcWRKY31 that located in nucleus (Bar = 50 µm; Notes: blue and green color dots indicate the nucleus of cells under microscope).

Figure 2 .
Figure 2. Bioinformatics analysis of AcWRKY31.(A) Gene structure of AcWRKY31.(B) Phylogenetic tree of AcWRKY31 and its homologous genes in Arabidopsis and rice.The unrooted phylogenetic tree was generated using MEGA 7.0 software with neighbor-joining procedure and the following pa-

Figure 2 .
Figure 2. Bioinformatics analysis of AcWRKY31.(A) Gene structure of AcWRKY31.(B) Phylogenetic tree of AcWRKY31 and its homologous genes in Arabidopsis and rice.The unrooted phylogenetic tree was generated using MEGA 7.0 software with neighbor-joining procedure and the following parameters: Poisson model, pairwise gap deletion, and 1000 bootstraps.Bar = 0.05 indicates the distance scale.(C) Multiple sequence alignment of AcWRKY31 and its homologous protein sequences (Note: the red dotted line refers to the WRKYGQK domain, and red triangles refer to the C 2 HC-type zinc finger motif).
Plants 2024, 13, x FOR PEER REVIEW 6 of 24 rameters: Poisson model, pairwise gap deletion, and 1000 bootstraps.Bar = 0.05 indicates the distance scale.(C) Multiple sequence alignment of AcWRKY31 and its homologous protein sequences (Note: the red dotted line refers to the WRKYGQK domain, and red triangles refer to the C2HC-type zinc finger motif).

Figure 3 .
Figure 3. Transformation and identification process of overexpression of AcWRKY31 in pineapple; (A) Agrobacterium transformation in pineapple callus; (B) Infected callus in 1st selection medium; (C) Plant regeneration from resistant callus in normal medium; (D) Plant regenerated callus in 2nd selection medium; (E) Resistant pineapple plantlets in normal medium; (F) Plantlets that died and changed color in 3rd selection medium; (G) Completed and resistant pineapple plantlets after undergoing selection three times; (H) Confirmation of positive transgenic pineapple by PCR method; (I) The expression level of AcWRKY31-OE lines and WT pineapple by RT-qPCR analysis.The error bars indicate ±SD (n = 3), and the asterisks indicate the significant differences based on Student's ttest (** p < 0.01).

Figure 3 .
Figure 3. Transformation and identification process of overexpression of AcWRKY31 in pineapple; (A) Agrobacterium transformation in pineapple callus; (B) Infected callus in 1st selection medium;

Figure 4 .
Figure 4. Abiotic stress treatments on transgenic pineapple; (A) Comparative phenotypic characters of WT and transgenic pineapple after 3 weeks of salt treatment; (B) Comparative phenotypic characters of WT and transgenic pineapple after 3 weeks of mannitol treatment; (C,D) The expression level of salt-and drought-stress-related genes by RT-qPCR analysis in AcWRKY31-OE lines and WT pineapple under abiotic treatments.The error bars indicate ±SD (n = 3), and the asterisks indicate significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Figure 4 .
Figure 4. Abiotic stress treatments on transgenic pineapple; (A) Comparative phenotypic characters of WT and transgenic pineapple after 3 weeks of salt treatment; (B) Comparative phenotypic characters of WT and transgenic pineapple after 3 weeks of mannitol treatment; (C,D) The expression level of salt-and drought-stress-related genes by RT-qPCR analysis in AcWRKY31-OE lines and WT pineapple under abiotic treatments.The error bars indicate ±SD (n = 3), and the asterisks indicate significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Figure 5 .
Figure 5. Inoculation of Dysmicoccus brevipes (pineapple mealybugs) on pineapple leaves; (A) Comparison of infected leaves between WT and AcWRKY31-OE pineapple plants after 3 months of inoculation, bars = 1 cm; (B) Comparison of infected mealybugs and number of colonies on dorsiventral surfaces in both WT and transgenic leaves (Bar= 1 cm) (C) Statistical results of mealybugs' colonies on dorsiventral surfaces in both WT and transgenic lines (the error bars indicate ±SD (n = 3)) (D) Free-hand section of pineapple leaves to understand the cell arrangement on both surfaces (ue: upper epidermal layer, le: lower epidermal layer, ch: chlorenchyma cell, ws: water storage cell, vb: vascular bundle, and fb: fiber; Note: cell arrangements are the same in both WT and transgenic lines).The asterisks indicate significant differences (*** p < 0.001).

Figure 6 .
Figure 6.Transcriptomic analysis of WT and AcWRKY31-OE transgenic pineapple plants.(A) GO analysis of the upregulated genes; (B) KEGG pathway analysis for upregulated genes; (C) GO analysis of the downregulated genes; (D) KEGG pathway analysis for downregulated genes.

Figure 6 .
Figure 6.Transcriptomic analysis of WT and AcWRKY31-OE transgenic pineapple plants.(A) GO analysis of the upregulated genes; (B) KEGG pathway analysis for upregulated genes; (C) GO analysis of the downregulated genes; (D) KEGG pathway analysis for downregulated genes.

Figure 7 .
Figure 7. Salt and drought treatments in AcWRKY31-OE Arabidopsis; (A) Observation of germination rate, fresh weight, and root length of AcWRKY31-OE and WT Arabidopsis under NaCl treatment (bar = 1 cm); (B) Observation of germination rate, fresh weight, and root length of AcWRKY31-OE and WT Arabidopsis under mannitol treatment (bar = 1 cm); (C,D) The expression of abiotic-stress-related genes in the WT and AcWRKY31 transgenic Arabidopsis plants in response to salt and drought stresses.Statistical results of fresh weight and root length after 7 days; the error bars indicate ±SD (n = 3), and the asterisks indicate the significant differences based on Student's t-test (** p < 0.01, * p ˂ 0.05).

Figure 7 .
Figure 7. Salt and drought treatments in AcWRKY31-OE Arabidopsis; (A) Observation of germination rate, fresh weight, and root length of AcWRKY31-OE and WT Arabidopsis under NaCl treatment (bar = 1 cm); (B) Observation of germination rate, fresh weight, and root length of AcWRKY31-OE and WT Arabidopsis under mannitol treatment (bar = 1 cm); (C,D) The expression of abiotic-stress-related genes in the WT and AcWRKY31 transgenic Arabidopsis plants in response to salt and drought stresses.Statistical results of fresh weight and root length after 7 days; the error bars indicate ±SD (n = 3), and the asterisks indicate the significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Figure 8 .
Figure 8. Inoculation of pathogen treatment on AcWRKY31-OE Arabidopsis.(A) The infection of Sclerotinia sclerotiorum on WT and AcWRKY31-OE Arabidopsis lines for 24 h and relative plaque area after DAB staining (bar = 1 cm); (B) The statistical data of lesion area; (C) The expression level of diseaserelated genes by RT-qPCR in AcWRKY31-OE and WT plants.The error bars indicate ±SD (n = 3), and the asterisk indicates significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Figure 8 .
Figure 8. Inoculation of pathogen treatment on AcWRKY31-OE Arabidopsis.(A) The infection of Sclerotinia sclerotiorum on WT and AcWRKY31-OE Arabidopsis lines for 24 h and relative plaque area after DAB staining (bar = 1 cm); (B) The statistical data of lesion area; (C) The expression level of disease-related genes by RT-qPCR in AcWRKY31-OE and WT plants.The error bars indicate ±SD (n = 3), and the asterisk indicates significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Figure 8 .
Figure 8. Inoculation of pathogen treatment on AcWRKY31-OE Arabidopsis.(A) The infection of Sclerotinia sclerotiorum on WT and AcWRKY31-OE Arabidopsis lines for 24 h and relative plaque area after DAB staining (bar = 1 cm); (B) The statistical data of lesion area; (C) The expression level of diseaserelated genes by RT-qPCR in AcWRKY31-OE and WT plants.The error bars indicate ±SD (n = 3), and the asterisk indicates significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Figure 9 .
Figure 9. Inoculation of pathogen treatment on Atwrky53 Arabidopsis.(A) The infection of Sclerotinia sclerotiorum on WT, Atwrky53 mutant, Atwrky53 complementary lines, and Atwrky53p35S:AcWRKY31 Arabidopsis lines for 24 h and relative plaque area after DAB staining (bar = 1 cm); (B) The statistical data of lesion area; (C) The expression levels of disease-related genes by RT-qPCR in transgenic lines and WT plants.The error bars indicate ±SD (n = 3), and the asterisks indicate significant differences based on Student's t-test (** p < 0.01, * p < 0.05).

Plants 2024 , 24 Figure 10 .
Figure 10.A role model of AcWRKY31-OE under biotic and abiotic stresses.AcWRKY31 gene regulates enzyme-and stress-related genes and then negatively responds to salt and drought stresses while it positively responds to disease by regulating hormone-and disease-related genes.

Figure 10 .
Figure 10.A role model of AcWRKY31-OE under biotic and abiotic stresses.AcWRKY31 gene regulates enzyme-and stress-related genes and then negatively responds to salt and drought stresses while it positively responds to disease by regulating hormone-and disease-related genes.